Nitric oxide (NO) is involved in a variety of biological processes including, for example, vascular control, neuronal signaling, control of clotting, and modulation of inflammatory responses. Nitric oxide is synthesized in most cells of the body by the enzyme nitric oxide synthase (NOS). Several forms of nitric oxide synthases are known. In an inflammatory response it is believed that the production of NO is a key factor in determining the magnitude of the inflammatory response. The NO produced by cells involved in inflammatory process is produced by a form of nitric oxide synthases called inducible nitric oxide synthases (iNOS). Macrophage cells are an example of cells that produce iNOS.
By measuring the production of NO by cell cultures exposed to various stimuli it is possible to quantify the cells response to the stimuli and, accordingly, whether the stimuli will induce, for example, an inflammatory response and the magnitude of the response. Current methods for measuring the rate of production of NO by cell cultures require extracting the nitric oxide synthases from the cells and then measuring the activity of the extracted nitric oxide synthases in vitro. Typically, the nitric oxide synthases is extracted, the extracted nitric oxide synthases is contacted with the substrates arginine and excess O2, and the rate of formation of citrulline is used to assess nitric oxide synthases activity in vitro. The extraction process, however, can radically change the activity of the nitric oxide synthases. Thus, the assay may not measure the actual activity of nitric oxide synthases in the cells of the cell culture. Also the extraction process destroys the cells.
R. S. Lewis et al. in “Kinetic Analysis of the Fate of Nitric Oxide Synthesized by Macrophages In Vitro” in Journal of Biological Chemistry, 270, 29350-29355 (1995) discloses a process for measuring NO production by macrophage cells by adhering the macrophage cells to microbeads, suspending the beads in media in a sealed container with a stirring mechanism to encourage mixing, and measuring NO concentrations in the headspace gas by directly measuring the partial pressure of NO and the partial pressure of the stable end products of NO oxidation, namely NO2 and NO3. In the disclosed method most of NO made by cells is produced at a significant distance from the NO sensor and is oxidized to NO2 and NO3 as it diffuses through the media. Since the NO is oxidized to NO2 and NO3 before it reaches the NO sensor it is not detected by the NO sensor. Accordingly, it is imperative in the disclosed system that the partial pressures of the oxidation products NO2 and NO3 be measured as well as the partial pressure of NO, in order to assess total NO production. Simultaneously measuring the partial pressures of NO2, NO3, and NO, however, is more complicated and time consuming than directly measuring only the partial pressures of NO.
One prior art method for measuring cellular respiration involves culturing adherent cells on plates at the bottom of culture wells and then covering the cells with a layer of media in the usual way. Cellular respiration is then measured using a sensor placed at some point near the cells. Knowing the gradient for the partial pressure of O2 in the media covering the cells and assuming the media has no convective mixing, the diffusion equation is solved to determine cellular respiration. When the partial pressure of O2 is measured by an electrode, this system has been called the open-air method. A system that functions in a similar way is offered commercially by BD Biosciences. In the system of BD Biosciences the partial pressure of O2 is measured by a fluorescent complex at the bottom of the culture well. This approach results in some convective mixing in the media that leads to substantial errors in determining cellular respiration.
Furthermore, prior approaches to controlling the partial pressure of oxygen in adherent cell cultures have suffered from the problem that convection in the growth media surrounding the cells is difficult to control. As a result, the diffusion of oxygen from the headspace gas, through the growth media, and to the cells is highly variable. Because of the variability of diffusion the partial pressure of oxygen at the cellular level is different from the partial pressure of oxygen in the headspace and it is impossible to determine the partial pressure of oxygen at the cellular level. Furthermore, restricted diffusion in the growth media makes it impossible to rapidly change the partial pressure of oxygen at the cellular level because any rapid changes in the partial pressure of oxygen in the headspace are damped out by diffusion through the growth media.
In another prior art method, the cells are grown under the experimental conditions of interest and are then scraped off of the surface they are growing on and re-suspended in media. The media is then placed in a sealed chamber, air bubbles are removed, and the partial pressure Of O2 in the media is measured. The decay in partial pressure of O2 versus time is directly related to cellular respiration. Thus, in the prior art techniques, (1) the cells must be scraped off and suspended, which frequently activates or inactivates adherent cells; (2) complete removal of gas bubbles is difficult, even a tiny bubble can ruin the results; and (3) cellular respiration cannot be changing during the measurement, otherwise the decay rate will not be constant.